miR-145a-5p/Plexin-A2 promotes the migration of OECs and transplantation of miR-145a-5p engineered OECs enhances the functional recovery in rats with SCI.

BACKGROUND: Olfactory ensheathing cells (OECs) serve as a bridge by migrating at the site of spinal cord injury (SCI) to facilitate the repair of the neural structure and neural function. However, OEC migration at the injury site not only faces the complex and disordered internal environment but also is closely associated with the migration ability of OECs. METHODS: We extracted OECs from the olfactory bulb of SD rats aged <7 days old. We verified the micro ribonucleic acid (miR)-145a-5p expression level in the gene chip after SCI and OEC transplantation using quantitative reverse transcription (qRT)-polymerase chain reaction (PCR). The possible target gene Plexin-A2 of miR-145a-5p was screened using bioinformatics and was verified using dual-luciferase reporter assay, Western blot, and qRT-PCR. The effect of miR-145a-5p/plexin-A2 on OEC migration ability was verified by wound healing assay, Transwell cell migration assay, and immunohistochemistry. Nerve repair was observed at the injured site of the spinal cord after OEC transplantation using tissue immunofluorescence and magnetic resonance imaging, diffusion tensor imaging, and the Basso-Beattie-Bresnahan locomotor rating scale were further used for imaging and functional evaluation. RESULTS: miR-145a-5p expression in the injured spinal cord tissue after SCI considerably decreased, while Plexin-A2 expression significantly increased. OEC transplantation can reverse miR-145a-5p and Plexin-A2 expression after SCI. miR-145a-5p overexpression enhanced the intrinsic migration ability of OECs. As a target gene of miR-145a-5p, Plexin-A2 hinders OEC migration. OEC transplantation overexpressing miR-145a-5p after SCI can increase miR-145a-5p levels in the spinal cord, reduce Plexin-A2 expression in the OECs and the spinal cord tissue, and promote OEC migration and distribution at the injured site. OEC transplantation overexpressing miR-145a-5p can promote the repair of neural morphology and neural function. CONCLUSIONS: Our study demonstrated that miR-145a-5p could promote OEC migration by down-regulating the target gene Plexin-A2, and transplantation of miR-145a-5p engineered OECs was beneficial to enhance neural structural and functional recovery in SCI rats.

Single-cell transcriptomic analysis of normal and pathological tissues from the same patient uncovers colon cancer progression.

The aim of the present study was to elucidate the evolutionary trajectory of colon cells from normal colon mucosa, to adenoma, then to carcinoma in the same microenvironment. Normal colon, adenoma and carcinoma tissues from the same patient were analyzed by single-cell sequencing, which perfectly simulated the process of time-dependent colon cancer due to the same microenvironment. A total of 22 cell types were identified. Results suggest the presence of dominant clones of same cells including C2 goblet cell, epithelial cell subtype 1 (Epi1), enterocyte cell subset 0 (Entero0), and Entero5 in carcinoma. Epi1 and Entero0 were Co-enriched in antibacterial and IL-17 signaling, Entero5 was enriched in immune response and mucin-type O-glycan biosynthesis. We discovered new colon cancer related genes including AC007952.4, NEK8, CHRM3, ANO7, B3GNT6, NEURL1, ODC1 and KCNMA1. The function of TBC1D4, LTB, C2CD4A, AND GBP4/5 in T cells needs to be clarified. We used colon samples from the same person, which provide new information for colon cancer therapy.

Macrophage Extracellular Traps Exacerbate Secondary Spinal Cord Injury by Modulating Macrophage/Microglia Polarization via LL37/P2X7R/NF-κB Signaling Pathway.

Persistent inflammation in the secondary spinal cord injury (SCI) is an important reason for the failure of nerve repair, which is partly due to the continuous activation of local M1-like macrophage/microglia. It is reported that extracellular trap (ET) has been a new way of cell death, which can be released by macrophages and named macrophage extracellular trap (Met). Furthermore, it exists widely in the pathophysiological process of many diseases, but it has been rarely studied in the field of SCI. In this study, we constructed a spinal cord contusion model and assessed the function outcome of SCI rats. We used immunofluorescence, flow cytometry, and transmission electron microscope (TEM) to demonstrate the existence of Mets. Besides, some related experiments had also been employed to explore the relationship between Mets and M1 polarization of macrophage/microglia. We also performed Co-IP and Western blotting to reveal a new extracellular proinflammatory signal pathway. Finally, we made a linear regression analysis between the concentrations of specific markers of Mets in human serum and ASIA scores. Briefly, our results suggested that macrophages infiltrated in SCI area could induce macrophage/microglia to differentiate into M1-like cells by releasing Mets, which may be achieved partly through LL37-P2X37-NF-κB signal pathway. However, limiting Mets could effectively inhibit M1 polarization and promote function recovery. In addition, the concentrations of Met related proteins in human serum showed high correlation with ASIA scores and could be applied to reflect the severity of SCI. In conclusion, Mets may be a new target for SCI therapy and a promising index for SCI assessment.

[miRNA-181a-5p inhibits proliferation and migration of osteosarcoma cell line HOS by targeting HOXB4].

OBJECTIVE: To study the effects and mechanisms of miR-181a-5p on the proliferation, cycle and migration of HOS osteosarcoma cells. METHODS: Real-time quantitative PCR was used to detect the expression of miR-181a-5p and HOXB4 in osteoblast hFOB1.19 cell line and osteosarcoma cell lines (HOS, U2OS, MG63). miR-181a-5p mimics and miR-181a-5p inhibitors were respectively transfected into HOS cells by Lipofectamine 2000, and miR NC group was set as control group. CCK-8 method was used to detect the change in cell proliferation. Flow cytometry was used to detect the changes in cell cycles. Wound healing experiments and Transwell migration experiments were used to detect the changes in cell migration ability. The target gene of miR-181a-5p was predicted by Targetscan website and validated by Dual-luciferase reporter gene system and Western blot. RESULTS: Compared with osteoblast hFOB1.19, miR-181a-5p was low expressed in osteosarcoma cells HOS, U2OS, and MG63(P<0.05), while HOXB4 was high expressed in osteosarcoma cells HOS, U2OS, and MG63(P<0.05). Compared with the miR NC group, over expression of miR-181a-5p inhibited the proliferation and migration of osteosarcoma HOS cells, and the number of cells in S phase decreased(P<0.05). However, knockdown miR-181a-5p promoted the proliferation and migration of osteosarcoma HOS cells, the cells in S phase increased(P<0.05). Bioinformatics prediction and Dual-luciferase reporter gene system validate HOXB4 as a downstream target gene of miR-181a-5p(P<0.05). Western blot showed that miR-181a-5p over expression or knockdown significantly down-regulated or up-regulated HOXB4 expressions in the HOS cells respectively(P<0.05). CONCLUSION: miR-181a-5p is down expressed in osteosarcoma cells, and over-expression miR-181a-5p inhibits the proliferation, cell cycle and migration ability of osteosarcoma cells by targeting HOXB4.

miR-672-3p Promotes Functional Recovery in Rats with Contusive Spinal Cord Injury by Inhibiting Ferroptosis Suppressor Protein 1.

Aberrantly expressed microRNAs (miRNAs) after spinal cord injury (SCI) participate in diverse biological pathways and processes, including apoptosis, inflammation, oxidative stress responses, peroxidation, and ferroptosis. This study was aimed at exploring the mechanisms underlying miRNA-mediated ferroptosis in an SCI rat model. In the present study, a T10 weight-dropping SCI model was established and miRNA profiling was used to detect miRNA expression profiles post-SCI. Basso-Beattie-Bresnahan scores and inclined plane test, hematoxylin and eosin (HE) and Nissl staining, immunohistochemistry and immunofluorescence, western blotting, cell viability, and Annexin V/7-aminoactinomycin D (7-AAD) assays were used to evaluate locomotor activity, histological changes in the injured spinal cords, neuronal ferroptosis, ferroptosis suppressor protein 1 (FSP1) expression, and cell death, respectively. It was observed that many miRNAs were differentially expressed after SCI, and miR-672-3p, which increased significantly, was selected after cross-referencing with predicted target miRNAs. The luciferase reporter assay demonstrated that miR-672-3p downregulated FSP1, a glutathione-independent ferroptosis suppressor, by binding to its 3' untranslated region. Oxygen and glucose deprivation- (OGD-) treated PC12 and AGE1.HN cells were treated with miR-672-3p mimics or inhibitors in vitro. The effect of miR-672-3p mimics or inhibitor on OGD-PC12/AGE1.HN ferroptosis was evaluated by flow cytometry, immunohistochemistry, immunofluorescence, and western blotting. The miR-672-3p mimics promoted ferroptosis after SCI, whereas the miR-672-3p inhibitor inhibited this process. Rats with SCI treated with miR-672-3p mimics or inhibitor showed similar results in vivo. Furthermore, the ferroptosis-related changes caused by SCI or miR-672-3p were reversed by overexpression of FSP1 lentivirus in vivo and in vitro. These results indicated that sh-miR-672-3p exerted a neural restoration effect in vivo and in vitro by inhibiting ferroptosis via the FSP1 pathway.

Lithium promotes recovery after spinal cord injury.

Lithium is associated with oxidative stress and apoptosis, but the mechanism by which lithium protects against spinal cord injury remains poorly understood. In this study, we found that intraperitoneal administration of lithium chloride (LiCl) in a rat model of spinal cord injury alleviated pathological spinal cord injury and inhibited expression of tumor necrosis factor α, interleukin-6, and interleukin 1 ß. Lithium inhibited pyroptosis and reduced inflammation by inhibiting Caspase-1 expression, reducing the oxidative stress response, and inhibiting activation of the Nod-like receptor protein 3 inflammasome. We also investigated the neuroprotective effects of lithium intervention on oxygen/glucose-deprived PC12 cells. We found that lithium reduced inflammation, oxidative damage, apoptosis, and necrosis and up-regulated nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 in PC12 cells. All-trans retinoic acid, an Nrf2 inhibitor, reversed the effects of lithium. These results suggest that lithium exerts anti-inflammatory, anti-oxidant, and anti-pyroptotic effects through the Nrf2/heme oxygenase-1 pathway to promote recovery after spinal cord injury. This study was approved by the Animal Ethics Committee of Xi'an Jiaotong University (approval No. 2018-2053) on October 23, 2018.

Vitamin D3 mediates miR-15a-5p inhibition of liver cancer cell proliferation via targeting E2F3.

Vitamin D3 has been demonstrated to suppress the development and progression of liver cancer, but the mechanism is unclear. The effects of vitamin D3 and microRNA (miR)-15a-5p on liver cancer cells were investigated in the present study using MTT and colony formation assays, flow cytometry, western blotting and reverse transcription-quantitative PCR. A dual-luciferase reporter assay was performed to determine whether E2F transcription factor 3 (E2F3) was a target of miR-15a-5p. The effects of silencing the E2F3 gene expression in liver cancer cells were investigated using a small interfering RNA. Vitamin D3 suppressed liver cancer cell proliferation, induced apoptosis and increased miR-15a-5p expression. Treatment with the miR-15a-5p mimics significantly suppressed liver cancer cell proliferation compared with that of the controls. Bioinformatics analysis and a dual-luciferase reporter assay demonstrated that E2F3 was a target of miR-15a-5p and that silencing E2F3 inhibited liver cancer cell proliferation. Therefore, Vitamin D3 suppressed cell proliferation by miR-15a-5p-mediated silencing of E2F3 gene expression. These findings suggested a role for vitamin D3 and E2F3 targeting as potential novel liver cancer therapies.

Transplantation of sh-miR-199a-5p-Modified Olfactory Ensheathing Cells Promotes the Functional Recovery in Rats with Contusive Spinal Cord Injury.

MicroRNAs (miRNAs) function as gene expression switches, and participate in diverse pathophysiological processes of spinal cord injury (SCI). Olfactory ensheathing cells (OECs) can alleviate pathological injury and facilitate functional recovery after SCI. However, the mechanisms by which OECs restore function are not well understood. This study aims to determine whether silencing miR-199a-5p would enhance the beneficial effects of the OECs. In this study, we measured miR-199a-5p levels in rat spinal cords with and without injury, with and without OEC transplants. Then, we transfected OECs with the sh-miR-199a-5p lentiviral vector to reduce miR-199a-5p expression and determined the effects of these OECs in SCI rats by Basso-Beattie-Bresnahan (BBB) locomotor scores, diffusion tensor imaging (DTI), and histological methods. We used western blotting to measure protein levels of Slit1, Robo2, and srGAP2. Finally, we used the dual-luciferase reporter assay to assess the relationship between miR-199-5p and Slit1, Robo2, and srGAP2 expression. We found that SCI significantly increased miR-199a-5p levels (P < 0.05), and OEC transplants significantly reduced miR-199a-5p expression (P < 0.05). Knockdown of miR-199a-5p in OECs had a better therapeutic effect on SCI rats, indicated by higher BBB scores and fractional anisotropy values on DTI, as well as histological findings. Reducing miR-199a-5p levels in transplanted OECs markedly increased spinal cord protein levels of Slit1, Robo2, and srGAP2. Our results demonstrated that transplantation of sh-miR-199a-5p-modified OECs promoted functional recovery in SCI rats, suggesting that miR-199a-5p knockdown was more beneficial to the therapeutic effects of OEC transplants. These findings provided new insights into miRNAs-mediated therapeutic mechanisms of OECs, which helps us to develop therapeutic strategies based on miRNAs and optimize cell therapy for SCI.

EGR1 interacts with DNMT3L to inhibit the transcription of miR-195 and plays an anti-apoptotic role in the development of gastric cancer.

EGR1 regulates the expression of its downstream target genes and may exert different biological effects in different tumours. We found that the expression of EGR1 was increased in gastric cancer (GC), and silencing the expression of EGR1 promoted the apoptosis of GC cells. Moreover, overexpression of EGR1 repressed the apoptosis of GC cells. Bioinformatics analysis showed that EGR1 had binding sites at the upstream promoter region of miR-195; ChIP assays were applied to determine EGR1 occupancy of the miR-195 promoter. The RT-PCR results showed that EGR1 suppressed the expression of miR-195. The mechanism by which EGR1 acts as a transcriptional repressor is still unclear. Bioinformatics analysis showed that EGR1 may interact with DNMT3L. We confirmed that EGR1 and DNMT3L formed a complex, and EGR1 was an important player in the transcriptional control of miR-195. Overexpression of miR-195 inhibited proliferation and promoted apoptosis in GC cells. We found a well-matched miR-195 binding site at the AKT3 3'-UTR. Double luciferase reporter assays showed that AKT3 was a target of miR-195, and silencing AKT3 repressed cell proliferation and promoted apoptosis. Our results indicated EGR1 may interact with DNMT3L to inhibit the miR-195-AKT3 axis and regulate the GC cell apoptosis.

MicroRNA-1297 inhibits proliferation and promotes apoptosis in gastric cancer cells by downregulating CDC6 expression.

Gastric cancer (GC), one of the most common malignant tumors and the second most common leading cause of cancer-related death worldwide, is a biologically heterogeneous disease accompanied by various genetic and epigenetic alterations. However, the molecular mechanisms underlying this disease are complex and not completely understood. Increasing studies have shown that aberrant microRNA (miRNA) expression is associated with GC tumorigenesis and growth. MiR-1297 has been confirmed to be a cancer suppressor in diverse tumors in humans. However, to date, the function and mechanism of miR-1297 in GC have not been determined. Here, we found that the expression of miR-1297 was significantly reduced in GC tissues or GC cell lines compared with paracarcinoma normal tissue or normal cell lines. Exogenic overexpression of miR-1297 in GC cell lines can inhibit cell proliferation and colony formation and induce apoptosis, and inhibition of miR-1297 in GC cell lines can promote cell proliferation and colony formation, and reduce apoptosis in vitro. We further confirmed that miR-1297 acted as a tumor suppressor through targeting cell division control protein 6 (CDC6) in GC. Moreover, the inverse relationship between miR-1297 and CDC6 was verified in GC cell lines. Our results indicated that miR-1297 is a potent tumor suppressor in GC, and its antiproliferative and gene-regulatory effects are, in part, mediated through its downstream target gene, CDC6. These findings implied that miR-1297 might be used as a novel therapeutic target of GC.

miR-99b-3p is induced by vitamin D3 and contributes to its antiproliferative effects in gastric cancer cells by targeting HoxD3.

Vitamin D3 is known to have anticancer actions by affecting tumorigenesis including the cell cycle and cell apoptosis in gastric cancer (GC) cells; the genes including microRNAs (miRNAs) regulated by vitamin D3 signaling remain discovered. miR-99b-3p, the tumor suppressor gene, is not only decreased in GC tissues, but is also induced by vitamin D3 through the vitamin D receptor (VDR) binding on the promoter domain of miR-99b. Further study indicates that miR-99b-3p inhibits cell viability and induces cell arrest in the S-phase in GC cells, the direct target gene of miR-99b-3p is verified to be HoxD3, which is also overexpressed in GC cell lines. Overall, our results show that miR-99b-3p mediates the antiproliferative of vitamin D3 in GC cells and might hold promise for prognosis and therapeutic strategies for GC treatment.

MECP2 promotes the growth of gastric cancer cells by suppressing miR-338-mediated antiproliferative effect.

The methyl-CpG-binding protein 2 (MECP2), a transcriptional suppressor, is involved in gene regulation by binding to methylated promoters. We found that MECP2 is overexpressed in gastric cancer (GC), and that Mecp2 knockdown affects the growth of GC cells both in vitro and in vivo. MECP2 can directly bind to the methylated-CpG island of miR-338 promoter and suppress the expression of two mature microRNAs, namely, miR-338-3p and miR-338-5p. Furthermore, miR-338-5p can suppress GC cell growth by targeting BMI1 (B lymphoma Mo-MLV insertion region 1 homolog). We additionally found that decreased miR-338-5p expression in GC tissues, relative to normal tissues, was significantly negatively correlated with increased BMI1 expression. Silencing MECP2 can indirectly lead to reduced expression of P-REX2, which has been identified as the miR-338-3p target, as well as BMI1 and increasing expression of P16 or P21 both in vitro and in vivo. Altogether, our results indicate that MECP2 promote the proliferation of GC cells via miR-338 (miR-338-3p and miR-338-5p)-mediated antitumor and gene regulatory effect.

miR-302b suppresses cell invasion and metastasis by directly targeting AKT2 in human hepatocellular carcinoma cells.

MicroRNAs (miRNAs) have been shown to play essential roles in regulating the activity of human hepatocellular carcinoma (HCC) cells, thereby contributing to the suppression of invasion and metastasis. In this study, using gain and loss of function assays, we demonstrated that miR-302b was frequently down-regulated in clinical HCC specimens, as compared with 15 corresponding adjacent normal tissues. Overexpression of miR-302b suppressed HCC cell invasion and metastasis. Regulation of NF-κB and matrix metalloproteinase (MMP)-2 expression by miR-302b was mediated via AKT2 in SMMC-7721 cells. Silencing AKT2 produced effects similar to those of miR-302b overexpression, which included inhibiting SMMC-7721 cell invasion and metastasis and dereasing NF-κB and MMP-2 expression. Furthermore, overexpression of AKT2 attenuated the effects of miR-302b overexpression. Taken together, our findings indicate that miR-302b inhibits SMMC-7721 cell invasion and metastasis by targeting AKT2, suggesting that miR-302b might represent a potential therapeutic target for HCC intervention.

[Effect of lipid accumulation after spinal cord injury in autofluorescence intensity of injury site].

OBJECTIVE: To investigate the relationship between lipid accumulation and autofluorescence intensity of injury site after spinal cord injury (SCI), and explore whether CuSO4 can eliminate autofluorescence in the injury site after SCI. METHODS: Thirty six Wild Type mice at age of 8 to 12 weeks (weight 18 to 24 g) were randomly divided into normal control group (n=4) and SCI group (n=32). Respectively, 8 mice of SCI group were sacrificed randomly at 1 week, 2 weeks, 4 weeks and 8 weeks after injury. Frozen sections of spinal cord tissue with injury site at the center were made to observe autofluorescence under green channel of fluorescence microscope (Specimens of normal control group were taken from the same segment of the spinal cord, and fixed with 4% paraformaldehyde solution). Oil Red O staining was applied to visualize the lipid accumulation in the injury site, and correlation between lipid accumulation and autofluorescence intensity was analyzed. Furthermore, sections were incubated with CuSO4 buffer to eliminate autofluorescence, and CuSO4 concentration and incubation time was optimized. RESULTS: No obvious autofluorescence or lipid staining was found in normal spinal cord tissue sections. By contrast, autofluorescence appeared in the injury site of spinal cord sections, and the intensity increased with passing time after injury. Oil Red O staining showed that lipid accumulated in the injury site with passing time after injury as well, and the correlation between lipid accumulation and autofluorescence intensity was positive. After incubation with CuSO4 buffer, the autofluorescence in the injury site was significantly reduced, especially after optimizing CuSO4 concentration and incubation time. CONCLUSIONS: Lipid accumulation may play an important role to determine the autofluorescence intensity of injury site after SCI, and the autofluorescence intensity can be used as a simple index for evaluating lipid peroxidation damage. Optimized method of using CuSO4 can significantly reduce the autofluorescence in the injury site after SCI, which will be beneficial to the application of immunofluorescence staining technique in the study of SCI.

MicroRNA-302b Enhances the Sensitivity of Hepatocellular Carcinoma Cell Lines to 5-FU via Targeting Mcl-1 and DPYD.

MiR-302b is a member of miR-302-367 cluster. The miR-302-367 cluster played important roles in maintaining pluripotency in human embryonic stem cells (hESCs) and has been proved to be capable of suppressing cell growth in several types of cancer cell lines including Hepatocellular Carcinoma (HCC) Cell lines. However, the role that miR-302b plays in the 5-Fluorouracil (5-FU) sensitivity of HCC has not been known. This study showed that miR-302b could enhance the sensitivity to 5-FU in HCC cell lines and verified its two putative targeted genes responsible for its 5-FU sensitivity.

miRNA-99b-3p functions as a potential tumor suppressor by targeting glycogen synthase kinase-3ß in oral squamous cell carcinoma Tca-8113 cells.

Dysregulation of microRNAs (miRNAs) has been associated with carcinogenesis in oral squamous cell carcinoma (OSCC). In the present study, we investigated the expression and function of miR-99b-3p in human OSCC. We found that the expression levels of miR-99b-3p decreased in 21 clinical OSCC samples (84%). Furthermore, ectopic expression of miR-99b-3p inhibited OSCC cell proliferation by downregulating glycogen synthase kinase-3ß (GSK3ß), an miR-99b-3p' target gene, at the mRNA and protein levels, both in vitro and in vivo. Moreover, the silencing of GSK3ß recapitulated the cellular and molecular effects in a similar manner to the overexpression of miR-99b-3p, which included inhibition of OSCC cell proliferation and suppression of p65 (RelA) and G1 regulators (cyclin D1, CDK4 and CDK6) in vitro. Our data suggest that miR-99b-3p functions as a tumor suppressor in OSCC via GSK3ß downregulation.

miR-15b-5p induces endoplasmic reticulum stress and apoptosis in human hepatocellular carcinoma, both in vitro and in vivo, by suppressing Rab1A.

In human hepatocellular carcinoma (HCC), aberrant expression of miRNAs correlates with tumor cell proliferation, apoptosis, invasion, and migration by targeting downstream proteins. miR-15b levels are reported increased in HCC tissues; however, they negatively correlate to HCC recurrence. Our aim was to understand the reason for this phenomenon. We used the reverse transcription-polymerase chain reaction (RT-PCR) to measure miR-15b-5p expression in both HCC tissues and HCC cell lines. Our results were consistent with the report. Using bioinformatics and luciferase reporter assays, we identified Rab1A as a novel and direct target of miR-15b-5p. Inhibiting the function of Rab1A with shRab1A also inhibited the growth of HCC cells and induced endoplasmic reticulum stress (ERS) and apoptosis. Similarly, suppressing Rab1A by overexpression of miR-15b-5p also inhibited cell growth and induced ERS and apoptosis. Moreover, re-expression of Rab1A rescued the miR-15b-5p-induced ERS, apoptosis, and growth inhibition in HCC cells. In vivo assays were further performed to testify them. Taken together, our data suggest that miR-15b-5p induces ERS, apoptosis, and growth inhibition by targeting and suppressing Rab1A, acting as a tumor suppressor gene in HCC. This finding suggests a novel relation among Rabs, miRNAs, and apoptosis.

miR-145 mediates the antiproliferative and gene regulatory effects of vitamin D3 by directly targeting E2F3 in gastric cancer cells.

VitaminD3 signaling is involved in inhibiting the development and progression of gastric cancer (GC), while the active vitamin D metabolite 1-alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3)-mediated gene regulatory mechanisms in GC remain unclear. We found that miR-145 is induced by 1,25(OH)2D3 in a dose- and vitamin D receptor (VDR)-dependent manner in GC cells. Inhibition of miR-145 reverses the antiproliferative effect of 1,25(OH)2D3. Furthermore, miR-145 expression was lower in tumors compared with matched normal samples and correlated with increased the E2F3 transcription factor protein staining. Overexpression of miR-145 inhibited colony formation, cell viability and induced cell arrest in S-phase in GC cells by targeting E2F3 and CDK6. miR-145 inhibition consistently abrogates the 1,25(OH)2D3-mediated suppression of E2F3, CDK6, CDK2 and CCNA2 genes. Altogether, our results indicate that miR-145 mediates the antiproliferative and gene regulatory effects of vitamin D3 in GC cells and might hold promise for prognosis and therapeutic strategies for GC treatment.

MicroRNA-214 suppresses the proliferation of human hepatocellular carcinoma cells by targeting E2F3.

MicroRNAs (miRNAs) are important gene regulators that participate in tumorigenesis. Previous studies have implicated that miR-214 is a tumor suppressor that is capable of inhibiting human hepatocellular carcinoma (HCC) cell growth. However, the mechanism by which miR-214 suppresses tumor development remains unknown. In the present study, miR-214 was observed to suppress tumor proliferation by directly targeting E2F transcription factor 3 (E2F3) in HCC cells. Colony formation, cell cycle and proliferation assays were employed to study the tumor suppressor role of miR-214 in cell proliferation. In addition, western blotting and dual-luciferase reporter assays were used to evaluate whether E2F3 was a target of miR-214. The results of these analyses revealed that E2F3 was a novel target of miR-214. Furthermore, enhanced expression of miR-214 or silencing of E2F3 inhibited the proliferation of HCC SMMC-7721 cells. These findings suggest that miR-214 suppresses HCC growth by targeting E2F3, and may provide a novel approach for the treatment of human HCC.

MicroRNA-195 acts as a tumor suppressor by directly targeting Wnt3a in HepG2 hepatocellular carcinoma cells.

MicroRNAs (miRNAs) are a class of small, non­coding, endogenous RNAs that are important in tumor cell biological processes as they regulate gene expression. miR-195 has been demonstrated to be a tumor repressor in numerous types of human cancer. However, the mechanism by which miR­195 suppresses tumor development remains to be elucidated. The aim of this study was to investigate the effect of miR-195 on the biological functions of HepG2 hepatocellular carcinoma (HCC) cells and identify the association between miR-195 and Wnt3a in HCC. miR-195 mRNA expression levels in HCC tissues and cell lines were measured by reverse transcription polymerase chain reaction analysis. miR-195 function was measured with cell proliferation, cell cycle and apoptosis assays following transfection with miR­195 and anti­miR­195 sequences, and the respective controls. Luciferase reporter assay was used to determine whether Wnt3a was a target of miR-195. In addition, Wnt3a expression levels were determined in HCC cells using western blot analysis. The miR-195 expression levels were found to be reduced in HCC tissues and cell lines. miR-195 overexpression resulted in a reduction in cell proliferation. In addition, the overexpression of miR-195 in HCC cells induced G1 phase cell cycle arrest and promoted apoptosis. Furthermore, Wnt3a was demonstrated to be directly targeted by miR-195. These findings suggest that miR-195 is key in regulating cell proliferation, cell cycle and apoptosis through targeting Wnt3a. In addition, overexpression of miR-195 may be a potential therapeutic strategy in the treatment of HCC.